The kidney collecting system arises through branching morphogenesis of the ureteric bud (UB), a process that we have been able to replicate in vitro by culturing the isolated UB in the presence of a metanephric mesenchyme cell conditioned medium. Our purification of proteins from this conditioned medium during the previous funding period has demonstrated that multiple soluble factors regulate UB branching. Here we argue that the ability of these growth factors to generate clear distinctions between UB tips and stalks is critical for normal collecting system development. We therefore seek to identify the cellular and molecular mechanisms by which particular growth factors work to generate tips and stalks (SA1). Since we have found that different sets of growth factors give very distinct patterns of isolated UB morphogenesis--"fine branching (tips and stalks)," "stalk-centric" and "tip-centric" phenotypes-we will use these clearly different conditions to identify molecules and mechanisms that lead to tip and stalk specification. In the proposed studies, we plan to connect the growth factors we have identified to cell surface signaling (via GFRalpha1 and lipid rafts) and intracellular signaling pathways (via Rho/ROCK), as well as intracellular protein sorting pathways (especially those involving ER chaperones), necessary for appropriate expression of tip and stalk-specific gene products. The logic of these connections, based largely on work completed during the previous funding period, is developed in detail in the application. (SA1 techniques: isolated UB culture, density gradient fractionation, transfections, selective down regulation of mRNA, laser capture microdissection, microarrays). In addition, we have identified at least one remaining potent activity for UB branching in vitro and aim to purify and characterize it. (SA2 techniques: column chromatography, mass spectrometry, protein expression). Together, the proposed studies should provide a much more complete picture of the soluble factors that regulate UB branching and how they act at the cell surface and intracellularly to generate tips and stalks during collecting system development. The experiments are based on considerable preliminary data and follow directly from work completed during the previous funding period. We have proven expertise in the techniques necessary to complete the project.